This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and increased risk for breast cancer. This information is needed to define early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, and to identify new targets, and facilitate selection and monitoring of women, for breast cancer prevention. Four clinical and laboratory studies are being conducted. Clinical studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) Protocol 02-C-0144, Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer (DN Danforth, PI). c.) Comprehensive literature review of molecular changes in normal breast tissue at low risk or at high risk for breast cancer, in hyperplasia, and in atypical hyperplasia. Protocol 02-C-0077 studies and characterizes by ductal lavage and ductal endoscopy the ducts and ductal epithelium of women at normal risk and increased risk for breast cancer. Forty-three women have been studied, 32 high risk subjects (contralateral breast in women with ipselateral breast cancer) and 11 at normal risk. Cytologic atypia of ductal epithelial cells was identified and correlated with ductal endoscopic findings of architectural changes. Repeat ductal lavage and endoscopy was performed on all subjects with atypia to define the persistence of cellular and ductal changes. A new method for acquisition of ductal epithelial cellular samples for cytologic and molecular analysis was developed: ductal endoscopic sampling with brush sampling devices of normal epithelium. This provided significantly increased cellular yields and samples of > 90% pure ductal epithelial cells. Molecular characteristics, including numerical and structural chromosome abnormalities and gene expression, of ductal epithelial cells will be defined by gene expression profiling, comparative genomic hybridization, and DNA methylation studies on normal and high risk epithelial cellular samples. (Danforth DN et al. Jour Surg Oncol, 94:555-564, 2006). Protocol, 02-C-0144 (DN Danforth, PI) establishes a tissue and cell line repository from all major sites of normal breast tissue at increased risk for breast cancer, including the contralateral normal breast, tissue adjacent to a breast cancer, women with a strong family history of breast cancer (including BRCA1 and BRCA2 mutation carriers), a Gail model risk estimate of breast cancer of > 1.67%, or women with prior mediastinal irradiation for lymphoma. Tissues are processed to allow for a spectrum of molecular studies. Mortal epithelial, fibroblast and adipose cell lines are developed to allow for a wide range of phenotypic, metabolic, and molecular studies. Demographic data is collected for each subject, and all specimens stored in an NIH repository. A comprehensive review of the major molecular changes - numerical chromosomal changes, structural chromosome changes, and changes in expression of individual genes has been conducted for the four types of preneoplastic breast tissue: normal/benign breast tissue at low risk for breast cancer, normal/benign tissue at high risk, epithelial hyperplasia, and epithelial hyperplasia with atypia to define the early changes and their significance in breast carcinogenesis. Patterns of molecular changes have been correlated with risk and with progression between histologic/morphologic subtypes. The earliest molecular changes described are loss of heterozygosity and DNA methylation of tumor suppressor loci, present in morphologically normal low risk, and more frequently high risk breast tissue, while aneusomy is a later event and is first identified in high risk normal breast tissue. Gene amplification is an uncommon event in early preneoplastic tissues. This review also provides important correlation and direction to both clinical studies described above. Proliferation of normal breast epithelial cells at increased risk for breast cancer is considered critical both for clonal expansion and for the accumulation of genetic abnormalities. To understand regulation of this proliferation, the effects of two prominent stimulatory and risk factors for breast cancer, estradiol (E2) and insulin-like growth factor-1 (IGF-1) on normal and high risk breast epithelial cells was studied. IGF-1 was found to significantly enhance proliferation and cell cycle progression of these epithelial cells, and modulated its own signaling pathway, decreasing expression of its receptor IGF-1R, stimulating expression of IRS-1, and stimulating phosphorylation of both signaling proteins. Estradiol alone had no effect on proliferation, cell cycle progression, or on modulation of its own receptor pathway (ER&#945;, ER&#946;, or PR). However, in combination with IGF-1, E2 modulated multiple components of the IGF signaling pathway. Together, this indicates that IGF-1, by enhancing proliferation of normal and high risk breast epithelial cell proliferation, may play an important promotional role in early carcinogenesis. These findings also provide new evidence of that E2 acts synergistically with IGF-1 to regulate multiple metabolic processes in these breast epithelial cells. The role of these agents in carcinogenesis will be further defined by studies of their regulation of DNA damage repair and signaling in normal and increased risk epithelium